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Selbach Lab

Proteome Dynamics

Profile

Understanding how the genomic information is interpreted to yield a specific phenotype is perhaps the most important question in the post-genomic era. We are investigating this by analysing protein-protein interactions and posttranslational modifications.

Proteins are the central players in this process: First, they are the final product of most genes. Therefore, understanding gene expression control requires analysis of protein synthesis and degradation. Second, function and dysfunction of proteins is directly responsible for cellular phenotypes.

Studying protein function can therefore provide novel insights into biological processes in health and disease. We are using quantitative mass spectrometry-based proteomics as our central technology to study proteome dynamics on a global scale.

The lab is interested in two major questions. First, how is the genomic information processed to yield a specific proteome? To answer this question we are studying protein synthesis and degradation. Second, how do proteins that are expressed at a specific cellular condition affect the phenotype?

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Research

Proteins are the chief actors in almost every biological process. While we know a lot about the function of individual proteins there is little information about the system as a whole. Recent developments in mass spectrometry have dramatically improved the analytical power of this technology.

We are using mass spectrometry-based quantitative proteomics to investigate cellular signalling at the protein level on a global scale. Main areas of research are posttranscriptional regulation of gene expression by microRNAs, protein-protein interaction in the context of neurodegenerative diseases and in vivo quantitative proteomics.

Research Projects

Understanding how the genomic information is interpreted to yield a specific phenotype is perhaps the most important question in the post-genomic era. Proteins are central players in this process: On the one hand, they are the final product of most genes. On the other hand, proteins are also directly responsible for cellular phenotypes. Proteins thus represent the central link between the genome and the phenotype. We are using quantitative mass spectrometry-based proteomics as our central technology to investigate proteome dynamics on a global scale. The lab is interested in two major questions. First, how is the genomic information processed to yield a specific proteome? Second, how do proteins that are expressed at a specific cellular condition affect the phenotype? Answering the first question requires information about protein synthesis and degradation. Systematic analysis of protein-protein interactions and posttranslational modifications can help answering the second question.

Fig. 1: Different variants of SILAC for the analysis of cellular proteome dynamics.

Recently developed quantitative methods make it possible to obtain precise functional information and to monitor temporal changes in the proteome by mass spectrometry. In one approach, named SILAC (for stable-isotope labelling with amino acids in cell culture), cells are differentially labelled by cultivating them in the presence of either normal or a heavy isotope–substituted amino acids. Due to their mass difference, pairs of chemically identical peptides of different stable-isotope composition can be distinguished in a mass spectrometer. The ratio of intensities for such peptide pairs accurately reflects the abundance ratio for the corresponding proteins. SILAC can be used to quantify differences in steady-state protein levels (Fig. 1, left). In addition, pulsed SILAC can be employed to measure differences in protein synthesis (Fig. 1, middle). Finally, dynamic SILAC can reveal protein turnover (Fig. 1, right). Combined with high throughput mass spectrometry, the different variants of SILAC enable quantification of different aspects of proteome dynamics on a global scale.

Gene expression control

The four fundamental cellular processes involved in gene expression are transcription, mRNA degradation, translation and protein degradation. Each of these four steps is controlled by gene-regulatory events. So far, little is known about how the combined effect of all regulatory events shapes gene expression. The fundamental question of how genomic information is processed to obtain a specific cellular proteome is therefore still largely unknown. We are using metabolic pulse labelling approaches to comprehensively quantify gene expression. Protein turnover can be quantified using dynamic SILAC. Similarly, newly synthesized RNA can be labelled with nucleoside analogues. Mass spectrometry and next generation sequencing (in collaboration with the lab of Wei Chen) then allows us to quantify the absolute abundance of mRNAs and proteins and their half-lives in parallel. These data can then be used to calculate synthesis rates of mRNAs and proteins by mathematical modeling (collaboration with the group of Jana Wolf). Our results indicate that gene expression in mouse fibroblasts is predominantly controlled at the level of translation (Schwanhausser, 2011). Consistently, we find that translation is actively regulated during Schwann cell development in vivo (collaboration with the lab of Carmen Birchmeier, Sheean et al., 2014). Currently, we are investigating how the different levels of gene expression change upon perturbation. In addition, we are using pulsed SILAC (pSILAC) to directly quantify changes in protein synthesis. For example, we are studying the impact of microRNAs and RNA-binding proteins on protein production.

Protein-protein interaction

Proteins typically interact with other proteins to exert a specific cellular function. Identifying interaction partners therefore provides direct insights into protein function and can reveal disease mechanisms. We are using quantitative mass spectrometry and to analyze protein-protein interactions (Paul et al., 2011). This approach has two unique advantages. First, accurate quantification allows us to distinguish specific interaction partners from background contaminants with very high confidence. Second, quantification reveals how interactions change in response to perturbation. We are using this method to study how disease-associated mutations and cell signaling events affect protein-protein interactions. We are also investigating how cellular interaction partners of influenza A virus proteins differ between strains with different pathogenic potential.

In vivo quantitative proteomics: The SILAC zoo

Cell culture-based experiments cannot recapitulate all of the complex interactions among different cell types and tissues that occur in vivo. Small animal models such as worms and fruit flies are attractive alternatives that are extensively used in many areas of biomedical research, especially in genetics and development. We have extended the SILAC technology to Caenorhabditis elegans (collaboration with the lab of Nikolaus Rajewsky) and Drosophila melanogaster (Sury et al., 2010; Grun et al., 2014). Currently, we are using these models to study protein-protein interactions in vivo.

High performance liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) can rapidly identify many proteins in complex biological samples. Recently, quantitative methods have been developed which make it possible to obtain precise functional information and to monitor temporal changes in the proteome by MS. In one approach, named SILAC (for stable-isotope labeling with amino acids in cell culture), cells are differentially labeled by cultivating them in the presence of either normal or a heavy isotope–substituted amino acid, such as 13C-labeled arginine. Metabolic incorporation of the amino acids into the proteins results in a mass shift of the corresponding peptides. Due to their mass difference, pairs of chemically identical peptides of different stable-isotope composition can be distinguished in a mass spectrometer. The ratio of intensities for such peptide pairs accurately reflects the abundance ratio for the two proteins. Quantitative proteomics with SILAC has emerged as a very powerful approach to investigate signaling processes. We are using this technology as our central tool to address several challenging questions in cell signaling.

Equipment

Our lab is equipped with the latest instrumentation for high end mass spectrometry. We have three hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher):

These instruments combine excellent mass accuracy with a high dynamic range, fast sequencing speed and high sensitivity. Peptide separation is performed on-line by nanoflow liquid chromatography using Easy nLC-1200 systems. Our proteomics pipeline is completed by hard- and software for efficient data processing such as a MASCOT server (Matrixscience) and MaxQuant (Juergen Cox, Matthias Mann lab, Max-Planck-Institute of Biochemistry). In addition, we have a fully equipped molecular cell biology lab for all kinds of biological experiments.